1. Field of the Invention
The present invention concerns genetically engineered low oxygen affinity mutants of human hemoglobin and the use of the same to increase tissue oxygenation in a human patient or to replace hemoglobin in a human patient. One aspect of the present invention resides in providing more oxygen to tumors.
2. Background Information
Blood consists of plasma and cells floating within it. The cells comprise white blood cells (leukocytes), platelets and red blood cells (erythrocytes). Red blood cells contain a protein, hemoglobin, which imparts color. It is the hemoglobin (Hb) which is involved in the transport of oxygen and carbon dioxide and which plays a role in regulating blood pH.
Each molecule of hemoglobin comprises four smaller subunits, called polypeptide chains. These are the protein or globin parts of hemoglobin. A heme group, which is an iron-protoporphyrin complex, is associated with each polypeptide subunit and is responsible for the reversible binding of one molecule of oxygen. Normal adult hemoglobin is made up of two different kinds of polypeptide subunits. One is called the alpha chain containing 141 amino acid residues and the other is called the beta chain and contains 146 amino acid residues. Two of each kind of such polypeptide chains are arranged in the form of a truncated tetrahedron which has an overall shape of an ellipsoid.
There are almost 300 known mutations of hemoglobin. Mutations lead to changes in the amino acid structure of the polypeptide chains. The abnormal gene responsible for sickle-cell anemia causes the normal glutamic amino acid residue at position six of the beta chain to be substituted by a valine group. Few of the known hemoglobin mutants, however, cause disease. Most mutants contain a mixture of mutant and normal hemoglobin.
The reversible combination of hemoglobin and oxygen is represented by the following reaction: ##STR1##
The equilibrium constants for each step are not the same because an oxygen molecule on one heme group changes (increases) the affinity of the other hemes for additional oxygen molecules. This alteration in binding affinity during oxygenation is called heme-heme interaction or cooperativity. Because of this cooperativity, the relationship between the partial pressure of oxygen and the amount of oxygen bound to hemoglobin is represented by a sigmoid curve. It is customary to characterize this sigmoid curve by two parameters: P.sub.50, the partial pressure of oxygen at which half saturation of the hemoglobin takes place; and n, the so-called Hill coefficient, which is a measure of cooperativity and thus the sigmoid shape of the curve. The Hill coefficient can vary in value from 1.0, corresponding to a lack of cooperativity and a straight line relationship, to 4.0, maximum cooperativity and an extreme sigmoid shape.
The oxygen affinity of hemoglobin is influenced by multiple factors including pH and the presence of certain inorganic phosphate molecules, most importantly 2,3-diphosphoglycerate. The term stripped hemoglobin is used to refer to hemoglobin free of these modulating inorganic phosphates. Stripped normal human hemoglobin has a P.sub.50 value of approximately 10 torr and a Hill coefficient of 2.8 to 3.0. The decrease in oxygen affinity with decrease in pH (more acidic) is known as the Bohr effect. An extreme form of the Bohr effect, known as the Root effect is observed for certain fish hemoglobin for which the value of P.sub.50 at pH circa 6.5 may be as high as several atmospheres.
X-irradiation is an important modality in the treatment of solid tumors. Two-thirds of the biological damage produced by x-rays occurs indirectly and is mediated by the action of free radicals. Oxygen combines with the free radicals and "fixes" the lesion in the cell. Therefore a thoroughly oxygenated tumor will be more responsive to x-irradiation.
Most solid tumors are not uniformly oxygenated. About 10-20% of the cells in experimental tumors are hypoxic. This occurs, at least in part, because tumors outgrow their blood supply. If more oxygen could be delivered to a tumor, cell death would be greater and tumor curability would improve.